Spectrophotometer baseline

As shown in Figure B2.1, double-beam spectrophotometers automatically record the true absorbance by measuring log(IR/Is), thanks to a double compartment containing two cuvettes, one filled with the solution and one filled with the solvent. Because the two cuvettes are never perfectly identical, the baseline of the instrument is first recorded (with both cuvettes filled with the solvent) and stored. Then, the solvent of the sample cuvette is replaced by the solution, and the true absorption spectrum is recorded. 

Selected entries from Methods in Enzymology [vol, page(s)] Absorption Spectrophotometer Application, 24, 15-25 baseline compensation, 24, 8-10 computerized, 24, 19-25 light scattering, 24, 13-15 monochromator, 24, 4 photometer, 24, 5-8 recorder, 24, 8 sample compartment, 24, 5 single-beam, 24, 3-4 spectral characteristics, 24, 10-12 split-beam, 24, 3 stray light, 24, 12-13. 

Figure 5. (A) Protein-difference spectrum for the binding of cellobiose onto CBH I (7.6°C). The baseline (a) was recorded (double beam spectrophotometer) with 0.720 mM cellobiose in the measuring cuvette and 0.720 mM sucrose in the reference cuvette. The difference spectrum (b) was recorded after addition of 9.3 /iM CBH I to both cuvettes.

Mod i f 1 ed Mjymbrajne Viscometer Foi- the pulsed system a coil of tubing (the injection loop) was placed after the prefilter and liefore the membrane holder as shown in Figure P. Directional valves at each end of the loop controlled the flow path. Solvent or solution could be pumped directly to the UV to establish baseline absorbance or for calibration. To make P measurements the flow was directed through the membrane and then into the differential UV spectrophotometer. The flow could also be brought to the upstream portion of tlie membrane holder and then to the UV detector in an effort to measure the concentration at the membrane surface. 

Determination of protein concentration (unitbu) requires an absorbance spectrum to be recorded on a good quality spectrophotometer from 240 to 350 nm. Aromatic amino acid residues do not absorb above 320 nm, so the spectrum between 320 and 350 nm should be only marginally above baseline. The presence of turbidity will result in finite attenuance in this region that increases toward lower wavelengths. 

Many spectrophotometers will allow for a rapid baseline correction to zero by using baseline adjust. 

In vivo light absorption spectra (400-700 nm) were recorded with a Varian-Cary spectrophotometer equipped with an integrating sphere. Samples were taken to an approximate optical density of 0.1. Data were corrected for scattering by subtracting a baseline value as measured at 725 nm. The spectrally averaged, chlorophyll a specific absorption cross section (aph, m2 mg Chi a-1) was calculated as follows ... 

Bromides and Iodides. The absorption spectra of the gaseous rare-earth halides were measured with a Cary 14 H spectrophotometer. The experimental procedure has been described previously 11). In this study a double furnace was used, allowing the rare-earth halide vapor to be heated to a higher temperature than the solid or liquid and allowing a baseline determination at the temperature of interest. In addition, a 0 0.1 full scale optical density slidewire was employed with the Cary... 

IR Analysis. IR absorption spectra were determined on neat samples of shale oils to furnish data for estimates of various olefinic types of compounds. Samples were run on a high-resolution, double-beam grating spectrophotometer at 0.1-mm path length between KBr plates. Quantitative measurements were made using the cut and weigh method with baselines drawn from point to point of minimum absorption. 

Very often baseline problems are related to detector problems. Many detectors are available for HPLC systems. The most common are fixed and variable wavelength ultraviolet spectrophotometers, refractive index, and conductivity detectors. Electrochemical and fluorescence detectors are less frequently used, as they are more selective. Detector problems fall into two categories electrical and mechanical/optical. The instrument manufacturer should correct electrical problems. Mechanical or optical problems can usually be traced to the flow cell however, improvements in detector cell technology have made them more durable and easier to use. Detector-related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or decreased sensitivity. Some cells, especially those used in refractive index detectors, are sensitive to flow and pressure variations. Flow rates or backpressures that exceed the manufacturer s recommendation will break the cell window. Old or defective source lamps, as well as incorrect detector rise time, gain, or attenuation settings will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems. 

It is probably best to use a variable-wavelength UV-Vis spectrophotometer and not a fixed wavelength instrument. These detectors are very stable and have excellent sensitivity. We have obtained an excellent baseline and chromatograms when the detector was set on 0.003 AU full scale. The variable-wavelength feature is useful because detectors of this type can be used to take advantage of rather small differences in absorbance spectra of various ions. 

Record the baseline of the spectrophotometer with the pressurizing liquid in the cell this is usually water or n-heptane. 

---